Evaluation of PCR primers for early diagnosis of cytomegalovirus infection following liver transplantation.
The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the HindIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% (HindIII-X) to 20% (MIE gene); however, specificity was inversely related (HindIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When HindIII-X and EcoRI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the HindIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.
Mendez
J C
JC
Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.
Espy
M J
MJ
Smith
T F
TF
Wilson
J A
JA
Paya
C V
CV
D000914:Antibodies, Viral / Q000032:analysis / Q000276:immunology; D001706:Biopsy; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000821:virology; D016031:Liver Transplantation / Q000009:adverse effects*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D014764:Viral Proteins
D000914:Antibodies, Viral; D017931:DNA Primers; D004279:DNA, Viral; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D014764:Viral Proteins; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII